miR156 Controls Forage Yield and Other Traits in Alfalfa by Regulating SPL Genes

MicroRNA156 (miR156) regulates many aspects of plant development, including juvenile to adult transition, shoot branching, and flowering time by post-transcriptional silencing of members of the SQUAMOUS PROMOTER-LIKE (SPL) gene family. Here, we investigated miR156 function in Medicago sativa (alfalfa) by overexpressing a precursor of miR156 (MsmiR156), and investigating its effects on gene expression and important traits in this plant. Three (SPL6, SPL12 and SPL13) of the five predicted SPL genes encoded by the alfalfa genome contain miR156 cleavage sites and their transcript levels were reduced in transgenic alfalfa plants overexpressing miR156. These transgenic plants had reduced internode length and stem thickness, enhanced shoot branching, increased trichome density, a delay in flowering time, and elevated biomass production. Minor effects on sugar, starch, lignin and cellulose contents were also observed. Moreover, overexpression of miR156 caused an increase in root length, with no noticeable effect on nodulation. The multitude of traits affected by miR156 in alfalfa may be due to the many genes that are regulated by the three target SPLs. Our results show that the miR156/SPL system has strong potential as a tool to substantially improve quality and yield traits in alfalfa.